A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala. Issue 1 (December 2015)

• Record Type: Journal Article
• Authors: Wampande, Eddie; Hatzios, Stavroula; Achan, Beatrice; Mupere, Ezekiel; Nsereko, Mary; Mayanja, Harriet; Eisenach, Kathleen; Boom, W; Gagneux, Sebastien; Joloba, Moses
• Is Part Of: BMC infectious diseases. Volume 15:Issue 1(2015)
• Page Start: 1
• Page End: 10
• Publication Date: 2015-12
• Subjects: Single nucleotide polymorphism -- Lineages -- Long sequence polymorphism -- High-throughput; Communicable diseases -- Periodicals; Sexually Transmitted Diseases -- Periodicals; 616.905
• DOI: 10.1186/s12879-015-1121-7
• Languages: English
• ISSNs: 1471-2334

Abstract

Abstract Background Accurate and high-throughput genotyping ofMycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients. Method Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing. Results The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage. Conclusion The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.